Work performed during the previous funding periods indicates that intrathecal administration of NPY reduces the mechanical and thermal hypersensitivity associated with inflammation or nerve injury. The objective of the present application is to establish NPY as an endogenous pain modulator, and to identify the mechanisms underlying NPY-mediated inhibition of inflammatory or neuropathic pain. The central hypothesis is that tissue or nerve injury sensitizes the dorsal horn to the pain inhibitory actions of NPY receptor signaling. Our approach involves transgenic, biochemical, neurophysiological, anatomical, and behavioral methods to determine: the anti-allodynic actions of endogenous NPY (Aim 1); the effect of injury on NPY receptor coupling to G-proteins (Aim 2); the effect of injury on the inhibition by NPY of SP release and subsequent NK1 receptor (NK1) activation in the spinal cord (Aim 3A); and the effect of NPY on the activity of spinal neurons that express the NPY Y1 receptor (Aim 3B). Our long-term goal is to understand endogenous neuropeptidergic inhibition of spinal pain transmission, and to establish the therapeutic potential of NPY receptor agonists for chronic pain. AIM #1 tests the hypothesis that endogenous NPY tonically inhibits allodynia. To address the contribution of endogenous NPY to chronic pain, Aim #1 will utilize transgenic mice (NPYtet) that contain a doxycycline-regulated cassette (tetracycline trans-activator, tTA) at the promoter region of the npy locus. We describe data indicating that inducible NPY depletion increases behavioral signs of allodynia after tissue or nerve injury. These data led us to determine the effect of conditional NPY knockdown on the induction, maintenance, and reinstatement of chronic pain. We predict that NPY depletion will speed the development, increase the intensity, and extend the duration of inflammatory and neuropathic pain. AIM #2 tests the hypothesis that injury increases NPY receptor-G protein signaling in the dorsal horn. To determine whether and when injury increases NPY receptor-G protein coupling, we will evaluate Y1- and Y2-agonist-stimulated [35S GTP3S binding at multiple time points after inflammation or nerve injury. AIM #3 tests the hypothesis that injury increases NPY inhibition of spinal pain neurotransmission. Primary afferent terminals in dorsal horn express NPY receptors. Our preliminary data indicate that NPY acts at Y1 and Y2 receptors to reduce substance P release and subsequent NK1 activation (assessed with NK1 internalization) in spinal cord slices and in vivo. We predict that injury will increase these inhibitory actions. Because dorsal horn neurons express Y1, we will test for postsynaptic actions of NPY on SP-induced nociceptive behavior, NK1 internalization, and spinal neuron gene action (Fos) on Y1-expressing cells. New preliminary data with the selective neurotoxin NPY-saporin suggest that Y1-expressing cells in the dorsal horn facilitate neuropathic pain transmission. We predict that the activity of this population of spinal neurons will be decreased by NPY administration, and increased by NPY knockdown or Y1 / Y2 receptor antagonism.